The present invention relates to a hydrogen peroxide sensor for fluids, and applications therefore. In particular, the present invention relates to a sensor which permits continuous monitoring of hydrogen peroxide in fluids and use of the data from the sensor for various purposes, including predicting hypertension and other oxidative stress-related physical conditions.
Hydrogen peroxide is formed in several biological and environmental processes. Hydrogen peroxide can be found in natural water (e.g., sea water, rain water), where it is an important species in redox reactions, in industrial processes, including drinking water purification, where it is used as a disinfectant, and in biological tissues, including blood, as a result of enzymatic reactions. Direct detection of hydrogen peroxide is an important analytical task, and numerous techniques have been devised for measurement of hydrogen peroxide levels in fluids as indications of, for example, medical conditions, environmental quality, or the presence of pathogens in cells of both animals and plants. Superoxide radicals (O2xe2x88x92) in living tissue can be derived from many sources, such as activated granulocytes, endothelial cells, xanthine oxidase-catalyzed reactions, mitochondrial metabolism, and transition metal reactions with oxygen. Hydrogen peroxide (H2O2) can be produced from the dismutation of superoxide radicals catalyzed by the enzyme superoxide dimutase (SOD), from transition metal reactions with superoxide radicals, and from enzymes (e.g., glycollate oxidase and urate oxidase) which produce peroxide directly without first producing superoxide. The presence of antioxidants, including certain enzymes such as SOD and catalase, serves to limit the concentration of the reactive oxygen species in plasma and tissues. Therefore, either an increase in the production of free radicals and/or a decrease in antioxidants can cause oxidative stress, contributing to possible cardiovascular complications in animals. Similarly, oxygen free radicals may affect vascular resistance by inactivating nitric oxide (NO), thereby causing arteriolar vasoconstriction and elevation of peripheral hemodynamic resistance. Other conditions have also been associated with oxidative stress, including arthritis, acceleration of the progression of HIV to full-blown AIDS, and neurological diseases such as ALS.
The mortality of individuals with hypertension has been found to be more than double that of the normotensive population, with most of the deaths occurring suddenly. Untreated hypertension also predisposes individuals to end organ damage or failure, including cerebrovascular accident (e.g., intracranial hemorrhage, encephalopathy), myocardial infarction, renal failure, and retinal hemorrhage. The mechanisms that predispose individuals with elevated arterial pressure to develop vascular organ injury are only partially understood. Oxygen free radicals and related intermediates have been implicated in hypertension and may play a role by affecting vascular smooth muscle contraction and resistance to blood flow. In individuals with histories of conditions such as atherosclerosis, stroke and myocardial infarction, hypertension constitutes a risk factor.
Studies have shown that in persons with essential hypertension there exists not only reduced antioxidant enzyme and nitric oxide levels, but also an increase in the NADPH oxidase activity on neutrophil membranes. An increase in NADPH oxidase activity results in production of oxygen free radicals. Consequently, hypertensives (individuals experiencing increased systolic and diastolic blood pressures) have higher superoxide and hydrogen peroxide production by neutrophils than normotensive (individuals with normal blood pressure) controls. It has also been shown that hypertensive patients revert to normal free radical, antioxidant and nitric oxide levels after effective antihypertensive treatment.
A number of different techniques are known for measurement of oxygen free radicals and their intermediates. These methods include the use of electrodes, chemiluminescence, and fluorescence. All of the aforementioned methods are limited to measuring oxygen free radicals from stimulated neutrophils or deproteinized whole blood.
A new hydrogen peroxide sensing system for measuring hydrogen peroxide in plasma is disclosed in international PCT patent application PCT/US 98/19013 (filed Sep. 14, 1998). In this system the test sample of plasma from a fluid or fluid-containing material which is to be analyzed for hydrogen peroxide content is divided into two equal portions and a hydrogen peroxide oxidation sensor is inserted into each portion. An inhibitor for the enzyme catalase, such as sodium azide, is added to one of the portions to stabilize the hydrogen peroxide present. A quantity of catalase is added to the other portion to deplete any hydrogen peroxide present by catalyzing it to oxygen. Hydrogen peroxide oxidation of each portion at the respective sensor is then measured, along with background oxidation of any other oxidizable species in the sample. The signal from the sensor in the depleted hydrogen peroxide sample is subtracted from the signal from the stabilized hydrogen peroxide sample to eliminate the signals"" contributions from background oxidation, thus yielding a resultant signal which is representative of the amount of hydrogen peroxide production in the subject fluid or material.
While the system described in that PCT application is quite useful, it requires two separate portions of the plasma from the sample fluid or material as well as chemical treatment of each of the portions. Such a system is useful primarily in a laboratory where there are facilities for chemically treating the portions, and where supplies of the treating chemicals can be made available. It is not, however, particularly useful for analysis of samples in the field or where the treating chemicals are not conveniently available. It also does not account for the fact that either or both of the treating agents may affect other components of the samples so that the two samples may end up being different from each other with respect to more than just the hydrogen peroxide component. Further, since the treating chemicals or enzymes must be added to each sample, the device must be recalibrated for each run.
We have now developed a new sensor probe which can measure hydrogen peroxide content of a single sample of a fluid or fluid-containing material (such as blood, tissue, environmental water steams or industrial water streams) using two oxygen sensors whose electrodes are encased in specified membranes. Each sensor has an oxygen sensing electrode group and both groups are inserted into the single sample of fluid or fluid-containing material, so that both measure from a homogeneous source, preventing testing errors due to differential reactions with treating chemicals.
The electrode end of each sensor is surrounded by a hydrophobic membrane which prevents the transport of electrochemical poisons or interferents and isolates the electrodes and an electrolyte fluid surrounding the electrodes from the sample fluid. The hydrophobic membrane is permeable to oxygen but not to hydrogen peroxide. The hydrogen-peroxide-generated-oxygen (HPGO) sensor also is encased in a hydrophilic membrane which contains an enzyme such as catalase, peroxidase or other enzymes of a family which catalyzes the reaction of hydrogen peroxide to oxygen and water, namely: 
This hydrophilic membrane is permeable to both hydrogen peroxide and oxygen. It is positioned in series with the hydrophobic membrane, with the hydrophobic membrane disposed between the hydrophilic membrane and the electrodes of the first sensor.
Disposed within the contained volume or space formed by the inner (proximal) surface of each hydrophobic membrane and the electrodes of its respective sensor, is an electrolyte solution to provide for migration of oxygen or electric charge to the appropriate electrodes. The electrolyte will be a fluid which is chemically inert to oxygen.
In operation the concentration gradient of oxygen in the test sample causes its diffusion across the membrane or membranes surrounding each sensor. The hydrophobic, pore free membranes surrounding each sensor prevent the diffusion of hydrogen peroxide and electrolytic poisons or interferents. At the oxygen reference sensor which is encased by only the hydrophobic membrane, only sample (background) oxygen diffuses into the internal electrolyte fluid until its concentrations in the electrolyte and test sample are equal. At the HPGO sensor, however, the hydrogen peroxide encounters the hydrophilic membrane with its catalase content. Catalytic reaction of the hydrogen peroxide with the catalase within the membrane results in the generation of oxygen in excess over the background sample oxygen level and the depletion of the hydrogen peroxide, so that the total quantity of oxygen which diffuses toward the HPGO sensor out of the hydrophilic membrane and through the hydrophobic membrane comprises both the background sample oxygen and the oxygen reaction product from the catalase-catalyzed conversion of the hydrogen peroxide.
The oxygen content is each fluid electrolyte is then detected by the respective sensors and the signals of each of the oxygen sensors are sent to a summer, which cancels out (subtracts) the portion of each signal due to the equal background oxygen concentration seen by each sensor. The resultant difference signal output by the summer thus represents only the quantity of oxygen generated by the conversion of the hydrogen peroxide at the HPGO sensor, and thus is a measure of the hydrogen peroxide content of the sample itself. A suitable display or data collection device may receive the resultant signal and provide a visible readout representing the hydrogen peroxide content, or the device may collect the signal data in electronic form which can be subsequently stored, manipulated and recovered.
Thus, in a broad embodiment, the invention is of a sensing probe for quantitative determination of hydrogen peroxide present in a body of fluid or fluid-containing material, which comprises first and second oxygen sensors, each generating a signal proportional to oxygen content of a fluid electrolyte in contact with a respective sensor; first and second membranes disposed in series and separating the first oxygen sensor from the material, with the second membrane being disposed between the first membrane and the first oxygen sensor; a third membrane separating the second oxygen sensor from the material; the first membrane being permeable to hydrogen peroxide and oxygen, and having dispersed therethrough an immobilized enzyme which catalyzes the conversion of hydrogen peroxide to oxygen; the second and third membranes being hydrophobic and permeable to oxygen but not hydrogen peroxide; and a summer receiving oxygen-content-dependent signals from the first and second oxygen sensors and generating a resultant signal proportional to the difference between the oxygen-content-dependent signals, the difference being proportional to the concentration of hydrogen peroxide in the sample.
In another broad embodiment the invention is of a sensing probe for quantitative determination of hydrogen peroxide present in a body of fluid or fluid-containing material, which comprises a first oxygen sensor and a second oxygen sensor, each having electrode means for detecting the presence of oxygen in a fluid electrolyte in contact with the electrode means and for generating a signal proportional to the concentration of oxygen detected in the fluid electrolyte; a first membrane surrounding the electrode means of the first oxygen sensor, forming a barrier between the electrode means and the material and being permeable to oxygen and hydrogen peroxide, the first membrane having disposed therethrough an immobilized enzyme which catalyzes conversion of hydrogen peroxide to oxygen; a second membrane and a third membrane, the second membrane also surrounding the electrode means of the first sensor in series with the first membrane and being disposed between the first membrane and the electrode means, and the third membrane surrounding the electrode means of the second sensor and forming a barrier between the electrode means of the second sensor and the material, the second and third membranes being hydrophobic and permeable to oxygen but not hydrogen peroxide; a summer electrically connected to the first and second oxygen sensors and receiving a signals from each sensor proportional to the amount of oxygen detected by the electrode means of the sensor, the summer comprising comparison means for determining difference in value between a signal from the first oxygen sensor and a signal from the second oxygen sensor and transmitting a difference signal proportional to the difference in value to a receiver; and the receiver comprising conversion means for receiving the difference signal and converting it into a human- or machine-readable indication of the concentration of hydrogen peroxide in the material.
Also a part of the present invention is a method of making a quantitative determination of hydrogen peroxide present in a body of fluid or fluid-containing material, such as human or animal tissue or bodily fluid or an environmental or industrial fluid or fluid-containing material, which comprises placing the fluid or fluid containing material in operative contact with a sensor probe of this invention, and operating the sensor probe to generate the human- or machine-readable indication of concentration of the hydrogen peroxide in the material.
The sensor probe thus provides for simple and rapid determination of the hydrogen peroxide content of a sample of tissue, blood, other bodily fluid or environmental or industrial fluid at any convenient facility and without the necessity of having additional chemicals present. The probe can advantageously be used in hospital settings, intensive care units, rehabilitation units, field locations, industrial plants or other locations where assays of hydrogen peroxide may be necessary or helpful.